Transcription (genetics)
illustrating the growing primary transcripts. "Begin" indicates the 3' end of the DNA template strand, where new RNA synthesis begins; "end" indicates the 5' end, where the primary transcripts are almost complete.]] Transcription is the synthesis of RNA under the direction of DNA. Both nucleic acid sequences use the same language, and the information is simply transcribed, or copied, from one molecule to the other. DNA sequence is enzymatically copied by RNA polymerase to produce a complementary nucleotide RNA strand, called messenger RNA (mRNA), because it carries a genetic message from the DNA to the protein-synthesizing machinery of the cell. One significant difference between RNA and DNA sequence is the presence of U, or uracil in RNA instead of the T, or thymine of DNA. In the case of protein-encoding DNA, transcription is the first step that usually leads to the expression of the genes, by the production of the mRNA intermediate, which is a faithful transcript of the gene's protein-building instruction. The stretch of DNA that is transcribed into an RNA molecule is called a transcription unit. A transcription unit that is translated into protein contains sequences that direct and regulate protein synthesis in addition to coding the sequence that is translated into protein. The regulatory sequence that is before, or 5', of the coding sequence is called 5' untranslated region (5'UTR), and sequence found following, or 3', of the coding sequence is called 3' untranslated region (3'UTR). Transcription has some proofreading mechanisms, but they are fewer and less effective than the controls for copying DNA; therefore, transcription has a lower copying fidelity than DNA replication. As in DNA replication, transcription proceeds in the 5' → 3' direction. Only one of the two DNA strands is transcribed. This strand is called the template strand, because it provides the template for ordering the sequence of nucleotides in an RNA transcript. The DNA template strand is read 3' → 5' by RNA polymerase and the new RNA strand is synthesized in the 5'→ 3' direction. RNA polymerase binds to the 5' end of a gene (promoter) on the DNA template strand and travels toward the 3' end. Except for the fact that thymines in DNA are represented as uracils in RNA, the newly synthesized RNA strand will have the same sequence as the coding (non-template) strand of the DNA. For this reason, scientists usually refer to the DNA coding strand that has the same sequence as the resulting RNA when referring to the directionality of genes on DNA, not the template strand. Transcription is divided into 3 stages: initiation, elongation and termination. Pre-initiation In eukaryotes, RNA polymerase, and therefore the initiation of transcription, requires the presence of a core promoter sequence in the DNA. Promoters are regions of DNA that promote transcription and, in eukaryotes, are found at -30, -75, and -90 base pairs upstream from the transcription start site (abbreviated to TSS). Core promoters are sequences within the promoter that are essential for transcription initiation. RNA polymerase is able to bind to core promoters in the presence of various specific transcription factors. The most characterized type of core promoter in eukaryotes is a short DNA sequence known as a TATA box, found 25-30 base pairs upstream from the TSS. The TATA box, as a core promoter, is the binding site for a transcription factor known as TATA-binding protein (TBP), which is itself a subunit of another transcription factor, called Transcription Factor II D (TFIID). After TFIID binds to the TATA box via the TBP, five more transcription factors and RNA polymerase combine around the TATA box in a series of stages to form a preinitiation complex. One transcription factor, Transcription factor II H, has two components with helicase activity and so is involved in the separating of opposing strands of double-stranded DNA to form the initial transcription bubble. However, only a low, or basal, rate of transcription is driven by the preinitiation complex alone. Other proteins known as activators and repressors, along with any associated coactivators or corepressors, are responsible for modulating transcription rate. Initiation Unlike DNA replication, transcription does not need a primer to start. RNA polymerase simply binds to the DNA and, along with other cofactors, unwinds the DNA to create an initiation bubble so that the RNA polymerase has access to the single-stranded DNA template. In bacteria, transcription begins with the binding of RNA polymerase to the promoter in DNA. The RNA polymerase is a core enzyme consisting of five subunits: 2 α subunits, 1 β subunit, 1 β' subunit, and 1 ω subunit. At the start of initiation, the core enzyme is associated with a sigma factor (number 70) that aids in finding the appropriate -35 and -10 basepairs downstream of promoter sequences. Transcription initiation is far more complex in eukaryotes and archaea, the main difference being that eukaryotic polymerases do not recognize directly their core promoter sequences. In eukaryotes, a collection of proteins called transcription factors mediate the binding of RNA polymerase and the initiation of transcription. Only after certain transcription factors are attached to the promoter does the RNA polymerase bind to it. The completed assembly of transcription factors and RNA polymerase bind to the promoter, called transcription initiation complex. In prokaryotes RNA Polymerase bind the mRNA and then forms a "closed complex". This complex is unwound to create the open complex which has melted DNA from -12 to +2. Elongation One strand of DNA, the template strand (or non-coding strand), is used as a template for RNA synthesis. As transcription proceeds, RNA polymerase traverses the template strand and uses base pairing complementarity with the DNA template to create an RNA copy. Although RNA polymerase traverses the template strand from 3' → 5', the coding (non-template) strand is usually used as the reference point, so transcription is said to go from 5' → 3'. This produces an RNA molecule from 5' → 3', an exact copy of the coding strand (except that thymines are replaced with uracils, and the nucleotides are composed of a ribose (5-carbon) sugar where DNA has deoxyribose (one less oxygen atom) in its sugar-phosphate backbone). Unlike DNA replication, mRNA transcription can involve multiple RNA polymerases on a single DNA template and multiple rounds of transcription (amplification of particular mRNA), so many mRNA molecules can be produced from a single copy of a gene. This step also involves a proofreading mechanism that can replace incorrectly incorporated bases. Prokaryotic elongation starts with the "abortive initiation cycle". During this cycle RNA Polymerase will synthesize mRNA fragments 2-12 nucleotides long. This continues to occur until the σ factor rearranges, which results in the transcription elongation complex (which gives a 35 bp moving footprint). The σ factor is released before 80 nucleotides of mRNA are synthesized. Termination Bacteria use two different strategies for transcription termination: in Rho-independent transcription termination, RNA transcription stops when the newly synthesized RNA molecule forms a G-C rich hairpin loop, followed by a run of U's, which makes it detach from the DNA template. In the "Rho-dependent" type of termination, a protein factor called "Rho" destabilizes the interaction between the template and the mRNA, thus releasing the newly synthesized mRNA from the elongation complex. Transcription termination in eukaryotes is less well understood. It involves cleavage of the new transcript, followed by template-independent addition of A''s at its new 3' end, in a process called polyadenylation. Measuring and detecting transcription Transcription can be measured and detected in a variety of ways: *Northern blot* *RNase protection assay *RT-PCR* *In vitro transcription *In situ hybridization *DNA microarrays* (* These methods measure total RNA levels which often differ) Transcription factories Active transcription units are clustered in the nucleus, in discrete sites called ‘transcription factories’. Such sites could be visualized after allowing engaged polymerases to extend their transcripts in tagged precursors (Br-UTP or Br-U), and immuno-labeling the tagged nascent RNA. Transcription factories can also be localized using fluorescence in situ hybridization, or marked by antibodies directed against polymerases. There are ~10,000 factories in the nucleoplasm of a HeLa cell, among which are ~8,000 polymerase II factories and ~2,000 polymerase III factories. Each polymerase II factory contains ~8 polymerases. As most active transcription units are associated with only one polymerase, each factory will be associated with ~8 different transcription units. These units might be associated through promoters and/or enhancers, with loops forming a ‘cloud’ around the factory. History A molecule which allows the genetic material to be realized as a protein was first hypothesized by Jacob and Monod. RNA synthesis by RNA polymerase was established ''in vitro by several laboratories by 1965; however, the RNA synthesized by these enzymes had properties that suggested the existence of an additional factor needed to terminate transcription correctly. In 1972, Walter Fiers became the first person to actually prove the existence of the terminating enzyme. Roger D. Kornberg won the 2006 Nobel Prize in Chemistry "for his studies of the molecular basis of eukaryotic transcription". Reverse transcription Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA. HIV has an RNA genome that is duplicated into DNA. The resulting DNA can be merged with the DNA genome of the host cell. The main enzyme responsible for synthesis of DNA from an RNA template is called reverse transcriptase. In the case of HIV, reverse transcriptase is responsible for synthesizing a complementary DNA strand (cDNA) to the viral RNA genome. An associated enzyme, ribonuclease H, digests the RNA strand, and reverse transcriptase synthesises a complementary strand of DNA to form a double helix DNA structure. This cDNA is integrated into the host cell's genome via another enzyme (integrase) causing the host cell to generate viral proteins which reassemble into new viral particles. Subsequently, the host cell undergoes programmed cell death (apoptosis). Some eukaryotic cells contain an enzyme with reverse transcription activity called telomerase. Telomerase is a reverse transcriptase that lengthens the ends of linear chromosomes. Telomerase carries an RNA template from which it synthesizes DNA repeating sequence, or "junk" DNA. This repeated sequence of "junk" DNA is important because every time a linear chromosome is duplicated, it is shortened in length. With "junk" DNA at the ends of chromosomes, the shortening eliminates some repeated, or junk sequence, rather than the protein-encoding DNA sequence that is further away from the chromosome ends. Telomerase is often activated in cancer cells to enable cancer cells to duplicate their genomes without losing important protein-coding DNA sequence. Activation of telomerase can be part of the process that allows cancer cells to become immortal. References See also * Genetics * Molecular biology * Translation - process of decoding RNA to form polypeptides. * Splicing - process of removing introns from precursor messenger RNA (pre-mRNA) to make form messenger RNA (mRNA). * Reverse transcription - process viruses use to make DNA from RNA * Crick's central dogma - DNA is transcribed to RNA which is translated to polypeptides, never the other way around. Further reading * Lehninger Principles of Biochemistry, 4th edition, David L. Nelson & Michael M. Cox * Principles of Nuclear Structure and Function, Peter R. Cook * Essential Genetics, Peter J. Russell External links * Interactive Java simulation of transcription initiation. From Center for Models of Life at the Niels Bohr Institute. * Interactive Java simulation of transcription interference--a game of promoter dominance in bacterial virus. From Center for Models of Life at the Niels Bohr Institute. Category:Gene expression Category:Molecular biology